Single cell mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on cell populations at single-cell resolution. Datasets are generated with antibody panels (upwards of 40) in which each antibody is conjugated to a polymer chelated with a stable metal isotope, usually in the Lanthanide series of the Periodic Table. The antibodies recognize surface markers that delineate cell types and intracellular signaling molecules demarcating multiple cell functions such as apoptosis, DNA damage and cell cycle. By measuring all these parameters simultaneously, the signaling state of an individual cell can be measured at the network level. Given the capabilities of mass cytometry, and recognizing a growing international biomedical and pharmaceutical interest in its application to immunology, diagnostics, and drug development, this Project will extend the current features of mass cytometry to nearly double the number of assayable channels through the creation of novel chelator-isotope pairings as well as new nanodots for highly sensitive detection of surface molecules. Further, we will enable additional virtual channels that increase the number of parameters measured per cell to as many as 200 using advanced signal processing tools such as compressed sensing along with signature based labeling. Finally, we will adapt DNA based amplification techniques to allow for low expressed protein epitope events and RNA copy number measurements down to as few as 5 target antigens measured quantitatively per cell. As per prior years with our other mass Cytometry protocols and computational abilities, developing and perfecting these additional capabilities will greatly enable the other Projects within our U19 center and will serve as a basis for extending these capabilities to others in the biomedical community, including other U19 Centers.